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. 1988 Jul 25;16(14B):6779–6788. doi: 10.1093/nar/16.14.6779

Formation of covalent complexes between human O6-alkylguanine-DNA alkyltransferase and BCNU-treated defined length synthetic oligodeoxynucleotides.

T P Brent 1, J S Remack 1
PMCID: PMC338332  PMID: 3405749

Abstract

Repair of chloroethylnitrosourea (CENU)-induced precursors of DNA interstrand cross-links by O6-alkylguanine-DNA alkyltransferase (GAT or GATase) appears to be a factor in tumor resistance to therapy with this class of antineoplastic drugs. Since human GAT is highly specific for O6-guanine, yet the probable cross-link structure is N'-Guanine N3-cytosine ethane, rearrangement of the initial O6-guanine adduct via O6,N1ethanoguanine has been proposed. We suggested that GAT reaction with this intermediate would produce DNA covalently linked to protein through an ethane link from N1-guanine to the alkylacceptor site on GAT. In preliminary studies we demonstrated a covalent complex between GAT and carmustine (BCNU)-treated DNA by a precipitation assay method. We have now developed a method for isolating the reaction product of BCNU-treated synthetic 14-mer [32P]-labeled oligodeoxynucleotide and GAT using polyacrylamide gel electrophoresis. This approach can be used to characterize the adducts induced by CENUs that lead to complex formation with GAT. Results obtained to date are consistent with these adducts being precursors of DNA interstrand cross-links.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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